Field of the Invention
The invention relates to methods and compositions for determining mechanisms that impart an antimicrobial susceptibility phenotype of an organism.
Description of the Related Art
The antimicrobial susceptibility phenotype of an organism may be due to a variety of mechanisms including, for example, the expression of various enzymes that deactivate the antimicrobial of interest, or cell surface modifications that prevent entry of the antimicrobial.
Determination of a specific mechanism involved in an antimicrobial susceptibility phenotype can be important for epidemiological analysis and in determining an appropriate therapy. For example, if it is determined that a bacteria is non-susceptible to a β-lactam due to the expression of a β-lactamase enzyme, this information can be used to inform the possibility of administering a therapy that includes a β-lactamase inhibitor. If instead the organism is non-susceptible to the β-lactam and it is determined that the organism does not express the β-lactamase, this information can be used to determine that a β-lactamase inhibitor may not provide a suitable therapy.
Different technologies in the art may be used for determining the specific mechanisms involved in an antimicrobial susceptibility phenotype. For example, nucleic acid amplification techniques (NAA) such as polymerase chain reaction (PCR) can be used for detecting the presence of a gene such as a species-specific gene and a β-lactamase gene. However, such NAA techniques can only infer the possibility an organism expressing the gene. As such, NAA techniques can identify the presence of an identifying nucleic acid sequence or antimicrobial resistance gene of an organism but cannot determine if that gene is expressed and cannot determine the susceptibility of the organism to an antimicrobial.
Colorimetric assays have been developed for the detection of the expression of enzymes that inactivate an antimicrobial agent. The Carba NP test is one such example through which the presence of expressed carbapenemases can be detected due to a color change of a solution (Nordmann, P., L. Poirel, and L. Dortet, Rapid Detection of Carbapenemase-producing Enterobacteriaceae. Emerging Infectious Diseases, 2012. 18(9): p. 1503-1507). This test detects the presence of expressed carbapenemases and correlates to the carbapenem susceptibility of an organism. However, the assay cannot determine the identity of the organism and thus requires prior isolation of the organism and cannot determine susceptibility if the mechanism responsible is not enzymatic.
Automated systems exist that provide identification and susceptibility information in an integrated system. The combination of peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) for organism identification and automated microscopy for the monitoring of the organism's growth rate in the presence of antibiotics is one example of such a system (Metzger, S., R. A. Frobel, and W. M. Dunne Jr, Rapid simultaneous identification and quantitation of Staphylococcus aureus and Pseudomonas aeruginosa directly from bronchoalveolar lavage specimens using automated microscopy. Diagnostic Microbiology and Infectious Disease, 2014. 79(2): p. 160-165). However, this system lacks the ability to determine the mechanisms involved in the observed susceptibility phenotype.
Finally, traditional culture techniques may be used to identify organisms and determine the mechanisms involved in imparting a susceptibility phenotype. An example of such an assay is the Modified Hodge Test used for determining the presence of carbapenemases (Lee, K., et al., Modified Hodge and EDTA-disk synergy tests to screen metallo-β-lactamase-producing strains of Pseudomonas and Acinetobactet species. Clinical Microbiology and Infection, 2001. 7(2): p. 88-91). However, these techniques rely on observations derived from the antibiotic susceptibility phenotype of an organism and cannot detect the presence of a mechanism involved in the phenotype if the phenotype is not expressed.
The limitations of existing technologies thus requires information from multiple assays and systems in order to provide identification, phenotypic susceptibility determination, and determination of the mechanisms involved in imparting the susceptibility phenotype. Because of this, healthcare providers must purchase and operate different systems in order to acquire this information—a costly and complicated endeavor. Due to these limitations, there is a need for a single assay that provides identification, phenotypic susceptibility determination, and determination of the mechanisms involved in imparting the susceptibility phenotype.